Process for isolation of antibiotic av290

ABSTRACT

This disclosure describes a reversible complex of antibiotic AV290 with an alkali metal alkyl sulfate and a process for preparing same. The complex is useful as an animal feed supplement which significantly enhances the growth rate of animals and poultry.

[Jnited States Patent 1191 Waite Dec. 24, 1974 PROCESS FOR ISOLATION OFANTIBIOTIC [75] Inventor: Jack Peter Waite, Fareham, England [73]Assignee: American Cyanamid Company,

Stamford, Conn.

22 Filed: Apr. 27, 1973 21 Appl. No: 355,240

[52] US. Cl. 424/115, 424/123 [51] Int. Cl A61k 21/00 [58] Field 01Search 424/118, 123, 115

[56] References Cited UNITED STATES PATENTS 3,338,786 8/1967 Kunstmannet al 424/118 Primary Examiner-Jerome D. Goldberg Attorney, Agent, orFirm-Edward A. Conroy, Jr.

[57] ABSTRACT 5 Claims, N0 Drawings PROCESS FOR ISOLATION OF ANTIBIOTICAV290 BRIEF SUMMARY OF THE INVENTION This invention relates to a methodof recovering antibiotic AV290 from fermentation whole harvest mashescontaining it. More particularly, the process involves adding an alkalimetal alkyl sulfate (or mixtures thereof) either to the whole harvestmash or to the filtered fermentation liquor, and recovering the soprecipitated antibiotic-alkyl sulfate reversible complex (or mixture ofcomplexes) by any convenient means. The invention also relates to theuse of the so prepared complex in animal feed supplement compositionsfor enhancing the growth rate of animals such as poultry, swine, earlyweaned pigs, and ruminants such as cattle, sheep and goats.

DETAILED DESCRIPTION OF THE INVENTION Antibiotic AV290 is formed byfermentative biosynthesis during the cultivation under controlledconditions of Slreptomyces candidus NRRL 3218 and mutants thereof. Thepreparation and properties of antibiotic AV290 are set forth in US. Pat.No. 3,338,786 which is hereby incorporated by reference. The problem ofrecovering the antibiotic economically has been a serious one. In thepatent referred to above, adsorption on charcoal followed by elution andcolumn chromatography are employed. Such a process is not excessivelyexpensive when pure antibiotic is required for medical usage. However,when the antibiotic is to be used in animal feed supplement compositionsthe factor of cost is a very serious matter and there is, therefore, aneed for an inexpensive process of recovering the antibiotic for thispurpose.

The present invention deals with a process and in a more specific aspectalso with a product. The process involves the precipitation of theantibiotic either from the whole harvest mash or from the filteredfermentation broth by the addition of alkali metal alkyl sulfates. Thealkali metal alkyl sulfates operable in the novel process of the presentinvention may be represented by the following general formula:

wherein n is an integer from 9 to 17, inclusive, and M is sodium orpotassium. Typical such alkali metal alkyl sulfates which may beemployed are, for example, sodium decyl sulfate, potassium hendecylsulfate, sodium lauryl sulfate, potassium tridecyl sulfate, sodiummyristyl sulfate, potassium pentadecyl sulfate, sodium cetyl sulfate,potassium heptadecyl sulfate, and sodium octadecyl sulfate. Mixtures ofalkali metal alkyl sulfates may also be employed such as a mixture ofsodium hendecyl sulfate and potassium octadecyl sulfate; a mixture ofpotassium decyl sulfate and sodium heptadecyl sulfate; a mixture ofpotassium lauryl sulfate and potassium cetyl sulfate; a mixture ofsodium tridecyl sulfate, potassium myristyl sulfate, and sodiumpentadecyl sulfate; and the like. When mixtures of alkali metal alkylsulfates are employed, then a corresponding mixture of antibiotic-alkylsulfate complexes are obtained.

The novel process of the present invention provides almost completeremoval of the antibiotic activity from the fermentation mash or broth.Furthermore, the antibiotic-alkyl sulfate complex so obtained can beused without separation of the constituents in animal feed supplementcompositions, which is an important economic advantage. Therefore, inone of the aspects of the present invention the complex of antibioticAV290 and an alkali metal alkyl sulfate is included as a prod uct.

The product of the antibiotic and alkali metal alkyl sulfate has beenreferred to as a reversible antibioticalkyl sulfate complex. lts exactchemical nature has not been determined, but covalent bonding is notinvolved and the product is not a physical mixture. This complex,derived from the interaction of the antibiotic and an alkali metal alkylsulfate, is not necessarily combined in any limiting stoichiometry. Thechemical bonds are reversible since the antibiotic AV290 may berecovered from the complex by various means such adsorption on across-linked carboxymethyldextran gel column followed by elution withaqueous acid. While it is not intended to limit the present invention totheories of chemical constitution and the like, it seems probable thatthe complex of the present invention is sufficiently reversible so thatunder conditions of use in animal feed supplement compositions theantibiotic is set free upon ingestion.

As starting material for the novel process of the pres ent inventionthere may be employed the whole harvest mash obtained after completionof a fermentation with S. candidus NRRL 3218 or mutants thereof.Preferably, there is employed the fermentation liquor or broth which hasbeen clarified by removing the mycelia and other insolubles byfiltration. Diatomaceous earth or any other conventional filtration aidmay be used to as' sist in the filtration. ln-either case, the pH of thewhole mash or of the filtered broth is first adjusted to between 1.9 and2.1, preferably about 2.0, with an acid. Suitable acids for this purposemay be, for example, hydrochlo ric acid, sulfuric acid, trifluoroaceticacid, and the like, although even glacial acetic acid may be used. ThispH adjustment appears to be critical since below pH 1.9 there appears tobe degradation of antibiotic AV290 during drying of the filter cake evenat 40C. under vacuum. Then, an aqueous solution of an alkali metal alkylsulfate (or a mixture of alkali metal alkyl sulfates) is added slowly,with stirring, at ambient temperatures. The entire process of thepresent invention is preferably carried out at from about 15C. to about30C., conveniently at room temperature. The antibiotic and alkyl sulfateform a complex which is water insoluble and thus precipitates. Theprecipitated complex or, in the case of the whole mash, the precipitatedcomplex together with the fermentation mash solids, is then re moved byfiltration or centrifugation and dried. The products so obtained may bedried by (l) slurrying the wet solids in polar, water misciblenon-solvents such as acetone followed by filtration, rinsing andair-drying; or

' by (2) reslurrying the wet solids in water and freeze drying or spraydrying.

When the products of the present invention are thus carefully driedunder temperature conditions which do not degrade antibiotic AV290, theyare usually white to tan powders in the case of the alkyl sulfatecomplex. In the case of the alkyl sulfate complex associated with driedharvest mash solids, they are usually gray to brown powders or solids.In the dry form, these products are extremely stable, keeping withoutsignificant loss of antibiotic activity for considerable periods oftime. This long storage life is, of course, an important practicaladvantage.

ltis an advantage of the present inventionthat the amount of alkalimetal alkyl sulfate added to precipitate the complex with the antibioticis not particularly critical and no exact stoichiometric relations needbe followed. In general, the amount of alkali metal alkyl sulfaterequired to precipitate antibiotic AV290 from a whole harvest mash is1.52.5 grams per gram of AV290 activity in the mash. in the usual case,about 2.0 grams of alkali metal alkyl sulfate per gram of antibioticactivity in the mash is preferred. The AV290 content of the whole mashmay be readily determined by microbiological assay (after adjusting thepH to 8.0-9.0) as set forth in US. Pat. No. 3,338,786. The preferredmethod is an adaptation of the Staphylococcus aureus turbidimetric assayfor tetracycline that is described in Assay Methods of Antibiotics, aLaboratory Manual by Grove & Randall, Medical Encyclopedia, lnc. (1955),pages 48-52. The required amount of alkali metal alkyl sulfate is thenpreferably dissolved or suspended in a convenient quantity of water andthe aqueous solution or suspension is added to the whole mash asdescribed above. Any excess alkali metal alkyl sulfate present willmerely remain in solution upon filtration.

In general, the amount of alkali metal alkyl sulfate required toprecipitate antibiotic AV290 from a clarified liquor is about one gramper gram of AV290 activity in the clarified liquor. The higher level ofalkali metal alkyl sulfate required to precipitate AV290 from whole mashthan from clarified liquor is due to coprecipitation of other proteinmaterial present in the whole mash. Conveniently, the minimum amount ofalkali metal alkyl sulfate required to form the complex with theantibiotic in the clarified liquor from any particular fermentationbatch may be readily determined as follows. A sample (conveniently 50100ml.) of the fermentation whole harvest mash is taken and clarified byremoving the mycelia and other insolubles by filtration, preferably witha filter aid. The filtrate is then acidified to a pH of 1.9-2.1 withdilute aqueous mineral acid such as dilute hydrochloric acid, dilutesulfuric acid, dilute phosphoric acid, or the like. This solution isthen titrated with the particular aqueous solution of alkali metal alkylsulfate which is to be used until no further precipitate or turbidityforms. The amount of alkali metal alkyl sulfate solution for theclarified liquor of the fermentation batch is then calculated from thetiter of the sample taken, providing also for a slight excess.

This invention also relates to animal feed supplement compositionseffective in accelerating the growth rate ofanimals and poultry. Inrecent years the use of antibiotics in animal feeds for improving growthcharacteristics and efficiency of feed utilization has become ofconsiderable economic importance. In accordance with the presentinvention, the dried alkyl sulfate complex or the dried harvest mashsolids containing the alkyl sulfate complex, either along or incombination with suitable carriers, when added to an animal feed,

Large ruminants 350 Small ruminants 200 Non-ruminants Poultry Themilligrams per pound of antibiotic AV290 present in any particularsupplement composition of the present invention may be readilydetermined by bioassay (after adjusting the pH to 8.0-9.0) as set forthin US. Pat. No. 3,338,786. The preferred method is an adaptation of theStaphylococcus aureus turbidimetric assay for tetracycline that isdescribed in the manual "Assay Methods of Antibiotics, a LaboratoryManual" by D. C. Grove and W. A. Randall, Medical Encyclopedia lnc.(1955) pages 4852. From the potency data thus obtained, the pounds offeed supplement composition to be used per ton of feed may be readilycalculated.

A wide variety of carriers may be used in the preparation of the feedsupplement compositions of this invention containing the dried alkylsulfate complex or the dried harvest mash solids containing the alkylsulfate complex. Carriers suitable for use to make up the feedsupplement compositions including the following: soybean meal, alfalfameal, cotton seed oil meal, linseed oil meal, cornmeal, cane molasses,urea, bone meal, corncob meal, and the like. The carrier promotes auniform distribution of the complex in the finished feed into which thesupplement is blended. It thus performs an important function byensuring proper distribution of the complex throughout the feed.

For a clearer understanding of the invention, specific examples of itare set forth below. These examples are merely illustrative, and are notto be understood as limiting the scope and underlying principles of theinvention in any way.

EXAMPLE 1 Precipitation of Antibiotic AV290-Sodium Lauryl SulfateComplex from Clarified Liquor For these tests, a sample of technicalAV290 sulfate having a microbiological potency of 63.8% was cmployed.Portions of the AV290 sulfate were dissolved in water and the pH of thesolutions were adjusted to 2.0 with 15% H 50 Celite 545 (a diatomaceousearth) was added as a filter aid and 2% aqueous sodium lauryl sulfatesolution was added dropwise with stirring. The slurry was aged for 30minutes at room temperature and the solids were removed by filtrationand dried under vacuum at 40C. The spent filtrate and dry solids wereassayed microbiologically and the results are set forth in Table 1below.

EXAMPLE 2 Precipitation of Antibiotic AV290-Sodium Lauryl SulfateComplex from Whole Harvest Mash then one kg. of Dicalite 478 was addedas a filter aid. To the stirred mash was slowly added a solution of 240gm. of sodium lauryl sulfate in 3 liters of water. The treated mash wasthen aged with stirring for 1 hour at Samples of S. candidus shakerflask fermentation 5 room temperature and thesolids were removed y wholeharvest mashes were taken and the potency means ofafllter press. Thefilter cake was dried under thereof boosted by the addition of technicalAV290 Vacuum at e y there obiamed 1,973 sulfate. In the tests tabulatedin Table 11 below, 100 ml. of Product havmg a mlereblologlcal assay ofportions of mash were employed and the PH thereof 4.72%. This representsan antibiotic AV290 recovery was adjusted to 2.0 with aqueous sulfuricacid. from mash to dry Cake of 831%- Then, 3.0 grams of Dicalite 478 (adiatomaceous EXAMPLE 5 earth) were added and 2% aqueous sodium laurylsulfate solution was added dropwise with Stirring ThePrecipitationofAnt1b1ot1cAV290from Whole Harvest slurrys were aged for 3hours at room temperature and is Mash Usmg Sodlum Decyl Sulfate thesolids were removed by filtration and dried under A 400 ml. sample ofwhole shaker flask mash (microvacuum at 40C. The spent filtrates and drycakes were biological assay 4,590 y/ml.) was adjusted to pH 2.0 byassayed microbiologically and the results are set forth the addition of3.9 ml. of concentrated sulfuric acid, in Table 11 below. and then 5grams of Dicalite 478 were added as a filter TABLE 11 Test No. l 2 3 4 56 7 8 Mash assay in -y/ml. 1300 1300 1400 1400 1130 1130 975 975 Mashedboosted 1O y/ml. 5130 5130 5230 5230 4960 4960 R' t' r cj l li bsoN-wxvwo 0.78 0.78 1 15 1.53 1.61 2.02 3.08 4.10 71 of input inspentfiltrate 44.8 48.5 20.3 12.0 12.0 6.5 9.2 6.2 dry cake 49.9 50.7 76.785.6 87.6 90.5 88.6 88.7 AV290 balance 94.7 99.2 97.0 97.6 99.6 97.097.8 94.9

EXAMPLE 3 aid. To the sitrred mash were slowly added 9.68 grams Eff f HP f h C l f of Empicol 0137 (a 30.5% active solution of sodium 0 p i l iOmp ex mm decyl sulfate manufactured by Albright & Wilson 06 arvest asChemicals, Ltd.) diluted to 50 ml. with water. The

Samples of S. candidus fermentation whole harvest treated mash was agedh Surfing for 1 hour at mashes were boosted to 4,930 y/ml. by theaddition room temperature 9 the Sohds were removed y of technical AV290sulfate and the pH of the samples means of a Vacuum ofiltel The fillerCake was were adjusted with 15% aqueous sulfuric acid as tabuunderVacuum at 50 whereby there w e f lated in Table 111 below. Then, 3.0grams or Dicalite grams Of y product havmg a m q q g 478 were addedfollowed by 40 ml. ofa 2% aqueous soassay of 914%- Thls representsantlbllyene AV29O dium lauryl sulfate solution, added dropwise withstirrecovery from mash y Cake of i005 ring, to a C, H OSO Na:AV290 ratioof 1.62. The EXAMPLE 6 slurrys were aged for 3 hours at room temperatureand l the solids were removed by filtration and dried underpreelpltatlen of Aetlbleue AV2 90 from Whole Harvest vacuum at 40C. Thespent filtrates and dry cakes were Mash Usmg a Mlxtere of Sedlum cetylSulfate and assayed microbiologically and the results are set forthSodlum oleyl Sulfate in Table III belo A 400 ml. sample of whole shakerflask mash (micro- TABLE 111 Sample No. 1 2 3 4 5 pH of mash forprecipitation 16 1.8 2.0 2.2 2.4 '7: of input inspent filtrate 7,3 6.46.1 6.9 10.1 dry cake 55.3 71.6 92.8 83.3 64.2 Av290 balance 62.6 713.098.9 90.2 74.3

EXAMPLE 4 biological assay 4,590 y/ml.) was adjusted to pH 2.0 by

. the addition of 3.9 ml. of concentrated sulfuric acid, is g z sg g ggge x f i i gmg z Harvest and then 5 grams of Dicalite 478 were added as afilter g y aid. To the stirred mash were slowly added 13.51 grams A 30liter sample of pilot fermenter mash having a 65 of Empicol CHC 30 (a30% active paste of a mixture microbiological assay of 3,740 y/ml. wastreated as follows. The pH of the mash was adjusted to 2.0 by theaddition of 219 ml. of concentrated sulfuric acid, and

of sodium cetyl sulfate and sodium oleyl sulfate manufactured byAlbright & Wilson Chemicals, Ltd.) dispersed in water to give a totaldispersion volume of ml. The treated mash was then aged with stirringfor 1 hour at room temperature and the solids were removed by means of avacuum filter. The filter cake was dried under vacuum at 50C. wherebythere was obtained EXAMPLE 7 Growth Promoting Effect of AntibioticAV290-Lauryl Sulfate Complex Experimental Design The evaluation of agrowth promoter is based on average results obtained with a series offour 2 week feeding tests. 1n each test, there are three pens of 10chicks for each treatment. Test Materials AV290-lauryl sulfate complexwas tested at levels of 5, 10, and 40 ppm. of AV290 equivalent in thediet. Diet The basal diet used in these experiments was Broiler RationNo. 453 (Table V). Experimental diets were prepared by mixing theappropriate amount of test material with the basal diet in a Hobartmixer. All diets were fed ad libitum and feeding of experimental dietswas begun upon arrival of the chicks. Brooding Day-old Hubbard x ArborAcres broiler chicks were housed in electrically heated brooders in anairconditioned room (24C.) for 2 weeks. There were five male and fivefemale chicks per pen. Data Data recorded included initial weights,average sexed weights at 2 weeks, and 2-week feed consumption. ResultsAV290-lauryl sulfate complex improved weight gain and feed utilizationat all levels tested. The degree of response was related to the amountof antibiotic feed. Average 2 week results from all four tests are asfollows in Table [V with percent improvement over control in brackets.

TABLE V-Continued BROILER RATION NO. 453

Ingredient Stabilized fat 4.0 Dicalcium phosphate 1.2 Ground Limestone0.5 Sodium chloride 0.3 *Tra-Min No. 3 0.05 **Vitamin premix 5 Total100.00

Vitamin Premix for l-Ton Ingredient grams DL methionine 453.6 BHT 113.6Vitamin A (30.000 u/g) 100.0 Vitamin D1; (200,000 u/g) 5.0 Vitamin E(20.000 u/lb) 45.4 Riboflavin 4.0 Niacinamide 25.0 Ca. Pantothenate 8.0Vitamin K (menadione) 1.0 Parvo (1071). folic acid 13.0 Choline Chloride(507) 908.0 Proferm (20 mg B /lb) 227.0 Corn Oil 500 Fine ground cornTotal 4536.0

*Tra-Min No. 3

1 lh/Ton Element /1 Furnishes (ppm) Manganese 12.50 62.5 Iron 6.00 30.0Zinc 5.00 25.0 Copper (1.65 3.25 lodine 0.35 1.75 Cobalt 0.25 1.25Calcium min. 15.30 max. 1835 1 claim:

1. A process of recovering an antibiotic AV290-alkyl sulfate complexfrom a fermentation whole harvest mash containing antibiotic AV290 whichcomprises the steps of:

a. producing a fermentation liquor by filtering the whole harvest mash;

b. acidifying the fermentation liquor to a pH of from 1.9 to 2.1 with apharmacologically acceptable acid;

c. adding to the acidified liquor a complexing agent selected from thegroup consisting of compounds of the formula:

wherein n is an integer from 9 to 17, inclusive, and M is sodium orpotassium, and mixtures thereof until a sufficient amount of theantibiotic AV290- alkyl sulfate complex is imparted to said medium;

(1. removing the precipitated antibiotic AV290-alkyl sulfate complex;and

e. drying the antibiotic AV290-alkyl sulfate complex.

2. A process according to claim 1 wherein the complexing agent is sodiumdecyl sulfate.

3. A process according to claim 1 wherein the complexing agent is sodiumlauryl sulfate.

4. A process according to claim 1 wherein the complexing agent is amixture of sodium cetyl sulfate and sodium oleyl sulfate.

5. A dry antibiotic AV290-alkyl sulfate complex prepared in accordancewith the process of claim 1.

l l l

1. A PROCESS OF RECOVERING AN ANTIBIOTIC AV 290-ALKYL SULFATE COMPLEXFROM A FERMENTATION WHOLE HARVEST MASH CONTAINING ANTIBIOTIC AV290 WHICHCOMPRISES THE STEPS OF: A. PRODUCING A FERMENTIATION LIQUOR BY FILTERINGTHE WHOLE HARVEST MASH; B. ACIDIFYING THE FERMENTATION LIQUOR TO A PH OFFROM 1.9 TO 2.1 WITH A PHARMACOLOGICALLY ACCEPTABLE ACID; C. ADDING TOTHE ACIDIFIED LIQUID A COMPLEXING AGENT SELECTED FROM THE GROUPCONSISTING OF COMPOUNDS OF THE FORMULA:
 2. A process according to claim1 wherein the complexing agent is sodium decyl sulfate.
 3. A processaccording to claim 1 wherein the complexing agent is sodium laurylsulfate.
 4. A process according to claim 1 wherein the complexing agentis a mixture of sodium cetyl sulfate and sodium oleyl sulfate.
 5. A dryantibiotic AV290-alkyl sulfate complex prepared in accordance with theprocess of claim 1.